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Competent E. coli Cells

Competent E. coli Cells
(Morris Lab Protocol)

 

Reagents

Buffer 1: 30 mM KOAc, 100 mM RbC12, 10 mM CaCl2, 50 mM MnC12, 15% glycerol, pH 5.8

For 500 ml:

1.47 g KOAc (MW 98.14)
6.04 g RbC12 (MW 120.92)
0.74 g CaC12 (MW 147.02)
4.94 g MnC12-4H20 (MW 197.9)
75 ml glycerol
pH to 5.8 with dilute acetic acid.

Filter sterilize.

Buffer 2: 10 mM MOPS/KOH pH 6.5, 75 mM CaC12, 10 mM RbC12, 15% glycerol

For 100 ml:

0.21 g MOPS (MW 209.26)
1.10 g CaC12 (MW 147.02)
0.12 g RbC12 (MW 120.92)
15 ml glycerol
pH to 6.5 with 1 N KOH.

Filter sterilize.

Protocol:

Note: Keep buffers, tips, tubes, rotors, etc. ice cold

1) Streak desired E. coli strain on fresh LB plate. Grow overnight at 37°C.
2) Inoculate a single colony into 10 ml LB. Grow overnight, shaking at 37° C.
3) Inoculate about 1 ml into 200 ml LB in a 2 1 flask. Shake at 37°C until OD550 = 0.5.
4) Chill flask in ice-water 5 minutes.
5) Spin 5 minutes at 6,000 rpm in GS3 rotor.
6) Resuspend pellet in 80 ml ice cold Buffer 1.
7) Chill in ice-water 5 minutes.
8) Spin 5 minutes at 6,000 rpm in GS3 rotor.
9) Resuspend pellet in 8 ml ice cold Buffer 2.
10) Chill in ice-water 15 minutes.
11) Make 50, 100 and 200 ml aliquots in 1.5 ml Eppendorf tubes.
12) Flash-freeze in liquid nitrogen.
13) Store at -80oC.

 

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revised 6/19/1998 cla